Abstract/Project Summary Title: Mechanistic details of key integral-membrane enzymes for antimicrobial discovery The increasing number of antibiotic resistant strains of bacteria represents a significant threat to human health making the development of novel therapeutic strategies critical. The major component of the bacterial cell wall is the peptidoglycan layer that is a unique meshwork providing essential structural support; therefore, identifying ways to weaken this layer is an ideal antibiotic strategy. Currently, numerous therapeutics target the peptidoglycan synthesis pathway and their use has been extremely successful in medicine. The enzymes involved in the pathway have been extensively characterized except in the case of the membrane components. Most notable is MraY, an essential protein that catalyzes the first membrane step of peptidoglycan biosynthesis. MraY is an integral membrane protein that has resisted mechanistic understanding. There are a few known inhibitors of MraY, such as nucleoside antibiotics, demonstrating its potential as an antibiotic target; however, none has been advanced into clinical development programs. Our group has developed efficient total synthesis schemes for two of the most promising natural products, capuramycin and muraymycin, allowing for the identification of improved compounds (e.g. UT-324). In this proposal we describe purified samples of MraY suitable for structural studies with inhibitor molecules, enzymatic substrate mimics, the viral protein E, or MurG. Purified MraY enzymes are used in in vitro activity assays for characterizing homologs and various inhibitors. We expand our synthetic strategy to generate broadly targeted chemical libraries and then test these for enzyme and bacterial growth inhibitory activities. Combining these efforts in one program creates a feedback loop that strengthens structural and medicinal chemistry aspects of the projects. Excitingly, our current efforts toward MraY structural characterization have yielded a promising co-crystal that demonstrates a model of an inhibited complex at low resolution. This application describes our goal of developing a thorough mechanistic picture of MraY that will allow us to design and identify novel inhibitors as lead compounds for drug discovery. The aims are to 1) expand on targeted small molecule libraries to identify new MraY inhibitors and 2) develop a full mechanistic understanding using structural and biochemical studies of MraY in a variety of functionally relevant states. Our combined team of structural biologists and synthetic chemists provides an innovative approach to achieve these important goals.